Reovirus for the treatment of cellular proliferative disorders

ABSTRACT

Methods for treating proliferative disorders, by administering reovirus to a Ras-mediated proliferative disorder, are disclosed. The reovirus is administered so that it ultimately directly contacts ras-mediated proliferating cells. Proliferative disorders include but are not limited to neoplasms. Human reovirus, non-human mammalian reovirus, and/or avian reovirus can be used. If the reovirus is human reovirus, serotype 1 (e.g., strain Lang), serotype 2 (e.g., strain Jones), serotype 3 (e.g., strain Dearing or strain Abney), as well as other serotypes or strains of reovirus can be used. Combinations of more than one type and/or strain of reovirus can be used, as can reovirus from different species of animal. Either solid neoplasms or hematopoietic neoplasms can be treated.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation (and claims the benefit of priorityunder 35 USC §120) of U.S. application Ser. No. 11/807,915, filed May30, 2007, currently pending, which is a continuation application of U.S.application Ser. No. 10/916,777, filed Aug. 11, 2004, issued as U.S.Pat. No. 7,374,752, which is a divisional application of U.S.application Ser. No. 10/218,452, filed Aug. 15, 2002, issued as U.S.Pat. No. 6,811,775, which is a continuation application of U.S.application Ser. No. 09/594,343 filed Jun. 15, 2000, issued as U.S. Pat.No. 6,455,038, which is a continuation application of U.S. applicationSer. No. 09/256,824, filed on Feb. 24, 1999, issued as U.S. Pat. No.6,136,307. All applications to which the instant application claimspriority are herein incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention pertains to methods for treating ras-mediatedproliferative disorders in a mammal using reovirus.

REFERENCES

The following publications, patent applications and patents are cited inthis application:

-   U.S. Pat. No. 5,023,252-   Armstrong, G. D. et al. (1984), Virology 138:37;-   Aronheim, A., et al., (1994) Cell, 78:949-961-   Barbacid, M., Annu. Rev. Biochem., 56:779-827 (1987);-   Berrozpe, G., et al. (1994), Int. J. Cancer, 58:185-191-   Bischoff, J. R. and Samuel, C. E., (1989) Virology, 172:106-115-   Cahill, M. A., et al., Curr. Biol., 6:16-19 (1996);-   Chandron and Nibert, “Protease cleavage of reovirus capsid protein    mu 1 and mu 1C is blocked by alkyl sulfate detergents, yielding a    new type of infectious subvirion particle”, J. of Virology    72(1):467-75 (1998-   Chaubert, P. et al. (1994), Am. J. Path. 144:767; Bos, J. (1989)    Cancer Res. 49:4682-   Cuff et al., “Enteric reovirus infection as a probe to study    immunotoxicity of the gastrointestinal tract” Toxicological Sciences    42(2):99-108 (1998)-   Der, S. D. et al., Proc. Natl. Acad. Sci. USA 94:3279-3283 (1997)-   Dudley, D. T. et al., Proc. Natl. Acad. Sci. USA 92:7686-7689 (1995)-   Duncan et al., “Conformational and functional analysis of the    C-terminal globular head of the reovirus cell attachment protein”    Virology 182(2):810-9 (1991)-   Fields, B. N. et al. (1996), Fundamental Virology, 3rd Edition,    Lippincott-Raven;-   Gentsch, J. R. K. and Pacitti, A. F. (1985), J. Virol. 56:356;-   E. Harlow and D. Lane, “Antibodies: A laboratory manual”, Cold    Spring Harbor Laboratory (1988)-   Helbing, C. C. et al., Cancer Res. 57:1255-1258 (1997)-   Hu, Y. and Conway, T. W. (1993), J. Interferon Res., 13:323-328-   Laemmli, U. K., (1970) Nature, 227:680-685-   Lee. J. M. et al. (1993) PNAS 90:5742-5746;-   Lee, P. W. K. et al. (1981) Virology, 108:134-146-   Levitzki, A. (1994) Eur. J. Biochem. 226:1; James, P. W., et    al. (1994) Oncogene 9:3601; Bos, J. (1989) Cancer Res. 49:4682-   Lowe. S. W. et al. (1994) Science, 266:807-810;-   Lyon, H., Cell Biology, A Laboratory Handbook, J. E. Celis, ed.    Academic Press, 1994, p. 232-   Mah et al., “The N-terminal quarter of reovirus cell attachment    protein sigma 1 possesses intrinsic virion-anchoring function”    Virology 179(1):95-103 (1990)-   McRae, M. A. and Joklik, W. K., (1978) Virology, 89:578-593-   Millis, N E et al. (1995) Cancer Res. 55:1444;-   Mundschau, L. J. and Faller, D. V., (1992) J. Biol. Chem.,    267:23092-23098-   Nagata, L., et al., (1984) Nucleic Acids Res., 12:8699-8710-   Paul R. W. et al. (1989) Virology 172:382-385-   Raybaud-Diogene. H. et al. (1997) J. Clin. Oncology, 15(3):    1030-1038;-   Remington's Pharmaceutical Sciences, Mace Publishing Company,    Philadelphia Pa. 17^(th) ed. (1985)-   Robinson, M. J. and Cobb, M. H., Curr. Opin. Cell. Biol. 9:180-186    (1997);-   Rosen, L. (1960) Am. J. Hyg. 71:242;-   Sabin, A. B. (1959), Science 130:966-   Samuel, C. E. and Brody, M., (1990) Virology, 176:106-113;-   Smith, R. E. et al., (1969) Virology, 39:791-800-   Stanley, N. F. (1967) Br. Med. Bull. 23:150-   Strong, J. E. et al., (1993) Virology, 197:405-411;-   Strong, J. E. and Lee, P. W. K., (1996) J. Virol., 70:612-616-   Trimble, W. S. et al. (1986) Nature, 321:782-784-   Turner and Duncan, “Site directed mutagenesis of the C-terminal    portion of reovirus protein sigma1: evidence for a    conformation-dependent receptor binding domain” Virology    186(1):219-27 (1992);-   Waters, S. D. et al., J. Biol. Chem. 270:20883-20886 (1995)-   Wiessmuller, L. and Wittinghofer, F. (1994), Cellular Signaling    6(3):247-267;-   Wong, H., et al., (1994) Anal. Biochem., 223:251-258-   Yang, Y. L. et al. EMBO J. 14:6095-6106 (1995)-   Yu, D. et al. (1996) Oncogene 13:1359

All of the above publications, patent applications and patents areherein incorporated by reference in their entirety to the same extent asif each individual publication, patent application or patent wasspecifically and individually indicated to be incorporated by referencein its entirety.

STATE OF THE ART

Normal cell proliferation is regulated by a balance betweengrowth-promoting proto-oncogenes and growth-constrainingtumor-suppressor genes. Tumorigenesis can be caused by geneticalterations to the genome that result in the mutation of those cellularelements that govern the interpretation of cellular signals, such aspotentiation of proto-oncogene activity or inactivation of tumorsuppression. It is believed that the interpretation of these signalsultimately influences the growth and differentiation of a cell, and thatmisinterpretation of these signals can result in neoplastic growth(neoplasia).

Genetic alteration of the proto-oncogene Ras is believed to contributeto approximately 30% of all human tumors (Wiessmuller, L. andWittinghofer, F. (1994), Cellular Signaling 6(3):247-267; Barbacid, M.(1987) A Rev. Biochem. 56, 779-827). The role that Ras plays in thepathogenesis of human tumors is specific to the type of tumor.Activating mutations in Ras itself are found in most types of humanmalignancies, and are highly represented in pancreatic cancer (80%),sporadic colorectal carcinomas (40-50%), human lung adenocarcinomas(15-24%), thyroid tumors (50%) and myeloid leukemia (30%) (Millis, N Eet al. (1995) Cancer Res. 55:1444; Chaubert, P. et al. (1994), Am. J.Path. 144:767; Bos, J. (1989) Cancer Res. 49:4682). Ras activation isalso demonstrated by upstream mitogenic signaling elements, notably bytyrosine receptor kinases (RTKs). These upstream elements, if amplifiedor overexpressed, ultimately result in elevated Ras activity by thesignal transduction activity of Ras. Examples of this includeoverexpression of PDGFR in certain forms of glioblastomas, as well as inc-erbB-2/neu in breast cancer (Levitzki, A. (1994) Eur. J. Biochem.226:1; James, P. W., et al. (1994) Oncogene 9:3601; Bos, J. (1989)Cancer Res. 49:4682).

Current methods of treatment for neoplasia include surgery, chemotherapyand radiation. Surgery is typically used as the primary treatment forearly stages of cancer; however, many tumors cannot be completelyremoved by surgical means. In addition, metastatic growth of neoplasmsmay prevent complete cure of cancer by surgery. Chemotherapy involvesadministration of compounds having antitumor activity, such asalkylating agents, antimetabolites, and antitumor antibiotics. Theefficacy of chemotherapy is often limited by severe side effects,including nausea and vomiting, bone marrow depression, renal damage, andcentral nervous system depression. Radiation therapy relies on thegreater ability of normal cells, in contrast with neoplastic cells, torepair themselves after treatment with radiation. Radiotherapy cannot beused to treat many neoplasms, however, because of the sensitivity oftissue surrounding the tumor. In addition, certain tumors havedemonstrated resistance to radiotherapy and such may be dependent ononcogene or anti-oncogene status of the cell (Lee. J. M. et al. (1993)PNAS 90:5742-5746; Lowe. S. W. et al. (1994) Science, 266:807-810;Raybaud-Diogene. H. et al. (1997) J. Clin. Oncology, 15(3): 1030-1038).In view of the drawbacks associated with the current means for treatingneoplastic growth, the need still exists for improved methods for thetreatment of most types of cancers.

SUMMARY OF THE INVENTION

The present invention pertains to a method of treating a ras-mediatedproliferative disorder in a mammal selected from dogs, cats, sheep,goats, cattle, horses, pigs, humans and non-human primates, comprisingadministering to the proliferating cells an effective amount of one ormore reoviruses in the absence of BCNU under conditions which result insubstantial lysis of the proliferating cells. The reovirus may be amammalian reovirus or an avian reovirus. The reovirus may be modifiedsuch that the outer capsid is removed, the virion is packaged in aliposome or micelle or the proteins of the outer capsid have beenmutated. The reovirus can be administered in a single dose or inmultiple doses. The proliferative disorder may be a neoplasm. Both solidand hematopoietic neoplasms can be targeted.

Also provided is a method of treating a ras-mediated neoplasm in ahuman, comprising administering to the neoplasm a reovirus in an amountsufficient to result in substantial oncolysis of the neoplastic cells.The reovirus may be administered by injection into or near a solidneoplasm.

Also provided is a method of inhibiting metastasis of a neoplasm in amammal, comprising administering to the mammal a reovirus in an amountsufficient to result in substantial lysis of the neoplastic cells.

Also provided is a method of treating a suspected ras-mediated neoplasmin a mammal, comprising surgical removal of the substantially all of theneoplasm and administration of an effective amount of reovirus at ornear to the surgical site resulting in oncolysis of any remainingneoplastic cells.

Also provided is a pharmaceutical composition comprising a reovirus, achemotherapeutic agent and a pharmaceutically acceptable excipient withthe proviso that the chemotherapeutic agent is not BCNU.

Also provided is a pharmaceutical composition comprising a modifiedreovirus and a pharmaceutically acceptable excipient.

The methods and pharmaceutical compositions of the invention provide aneffective means to treat neoplasia, without the side effects associatedwith other forms of cancer therapy. Furthermore, because reovirus is notknown to be associated with disease, any safety concerns associated withdeliberate administration of a virus are minimized.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the molecular basis of reovirus oncolysis, inwhich the reovirus usurps the host cell Ras signaling pathway.

FIG. 2 is a graphic representation of the effects over time of active(open circles) or inactivated (closed circles) reovirus serotype 3(strain Dearing) on the size of murine THC-11 tumors grown in severecombined immunodeficiency (SCID) mice. The plotted values represent themean of the measurements with the standard error of the mean also shown.

FIG. 3 is a graphic representation of the effects over time of active(open circles) or inactivated (closed circles) reovirus serotype 3(strain Dearing) on the size of human glioblastoma U-87 xenografts grownin SCID mice. The plotted values represent the mean of the measurementswith the standard error of the mean also shown.

FIG. 4 is a graphic representation of the effects over time of active(open circles, open squares) or inactivated (closed circles, closedsquares) reovirus serotype 3 (strain Dearing) on the size ofinjected/treated (open and closed circles) or untreated (open and closedsquares) bilateral human glioblastoma U-87 xenografts grown in SCIDmice. The plotted values represent the mean of the measurements with thestandard error of the mean also shown.

FIG. 5 is a graphic representation of the effects over time of active(open circles) or inactivated (closed circles) reovirus serotype 3(strain Dearing) on the size of C3H transformed cell mouse tumors inimmunocompetent C3H mice.

FIG. 6 is a graphic representation of the effects over time of active(open circles, open squares) or inactivated (closed circles) reovirusserotype 3 (strain Dearing) on the size of C3H transformed cell mousetumors grown in immunocompetent C3H mice previously exposed (opensquares) or unexposed (open circles) to reovirus.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to methods of treating a ras-mediatedproliferative disorder in a mammal, by administering reovirus to theproliferating cells.

The name reovirus (Respiratory and enteric orphan virus) is adescriptive acronym suggesting that these viruses, although notassociated with any known disease state in humans, can be isolated fromboth the respiratory and enteric tracts (Sabin, A. B. (1959), Science130:966). The term “reovirus” refers to all viruses classified in thereovirus genus.

Reoviruses are viruses with a double-stranded, segmented RNA genome. Thevirions measure 60-80 nm in diameter and possess two concentric capsidshells, each of which is icosahedral. The genome consists ofdouble-stranded RNA in 10-12 discrete segments with a total genome sizeof 16-27 kbp. The individual RNA segments vary in size. Three distinctbut related types of reovirus have been recovered from many species. Allthree types share a common complement-fixing antigen.

The human reovirus consists of three serotypes: type 1 (strain Lang orT1L), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strainAbney, T3D). The three serotypes are easily identifiable on the basis ofneutralization and hemagglutinin-inhibition assays (Sabin, A. B. (1959),Science 130:966; Fields, B. N. et al. (1996), Fundamental Virology, 3rdEdition, Lippincott-Raven; Rosen, L. (1960) Am. J. Hyg. 71:242; Stanley,N. F. (1967) Br. Med. Bull. 23:150).

Although reovirus is not known to be associated with any particulardisease, many people have been exposed to reovirus by the time theyreach adulthood (i.e., fewer than 25% in children<5 years old, togreater than 50% in those 20-30 years old (Jackson G. G. and Muldoon R.L. (1973) J. Infect. Dis. 128:811; Stanley N. F. (1974) In: ComparativeDiagnosis of Viral Diseases, edited by E. Kurstak and K. Kurstak,385-421, Academic Press, New York).

For mammalian reoviruses, the cell surface recognition signal is sialicacid (Armstrong, G. D. et al. (1984), Virology 138:37; Gentsch, J. R. K.and Pacitti, A. F. (1985), J. Virol. 56:356; Paul R. W. et al. (1989)Virology 172:382-385) Due to the ubiquitous nature of sialic acid,reovirus binds efficiently to a multitude of cell lines and as such canpotentially target many different tissues; however, there aresignificant differences in susceptibility to reovirus infection betweencell lines.

As described herein, Applicants have discovered that cells which areresistant to reovirus infection became susceptible to reovirus infectionwhen transformed by a gene in the Ras pathway. “Resistance” of cells toreovirus infection indicates that infection of the cells with the virusdid not result in significant viral production or yield. Cells that are“susceptible” are those that demonstrate induction of cytopathiceffects, viral protein synthesis, and/or virus production. Resistance toreovirus infection was found to be at the level of gene translation,rather than at early transcription: while viral transcripts wereproduced, virus proteins were not expressed. Without being limited to atheory, it is thought that viral gene transcription in resistant cellscorrelated with phosphorylation of an approximately 65 kDa cell protein,determined to be double-stranded RNA-activated protein kinase (PKR),that was not observed in transformed cells. Phosphorylation of PKR leadto inhibition of translation. When phosphorylation was suppressed by2-aminopurine, a known inhibitor of PKR, drastic enhancement of reovirusprotein synthesis occurred in the untransformed cells. Furthermore, asevere combined immunodeficiency (SCID) mouse model in which tumors werecreated on both the right and left hind flanks revealed that reovirussignificantly reduced tumor size when injected directly into theright-side tumor; in addition, significant reduction in tumor size wasalso noted on the left-side tumor which was not directly injected withreovirus, indicating that the oncolytic capacity of the reovirus wassystemic as well as local.

These results indicated that reovirus uses the host cell's Ras pathwaymachinery to downregulate PKR and thus reproduce. FIG. 1 depicts theusurpation of the host cell Ras signalling pathway by reovirus. As shownin FIG. 1, for both untransformed (reovirus-resistant) and EGFR-, Sos-,or ras-transformed (reovirus-susceptible) cells, virus binding,internalization, uncoating, and early transcription of viral genes allproceed normally. In the case of untransformed cells, secondarystructures on the early viral transcripts inevitably trigger thephosphorylation of PKR, thereby activating it, leading to thephosphorylation of the translation initiation factor eIF-2α, and hencethe inhibition of viral gene translation. In the case of EGFR-, Sos-, orras-transformed cells, the PKR phosphorylation step is prevented orreversed by Ras or one of its downstream elements, thereby allowingviral gene translation to ensue. The action of Ras (or a downstreamelement) can be mimicked by the use of 2-aminopurine (2-AP), whichpromotes viral gene translation (and hence reovirus infection) inuntransformed cells by blocking PKR phosphorylation.

The implantation of human tumor cells into SCID mice is recognized as awell known model system for testing the effectiveness of variousanti-tumor agents in humans. It has previously been shown thatpharmaceuticals effective against human tumors implanted into SCID miceare predictive of their effectiveness against the same tumors in humans.

Based upon these discoveries, Applicants have developed methods fortreating ras-mediated proliferative disorders in mammals. Representativemammals include dogs, cats, sheep, goats, cattle, horses, pigs,non-human primates, and humans. In a preferred embodiment, the mammal isa human.

In the methods of the invention, reovirus is administered toras-mediated proliferating cells in the individual mammal.Representative types of human reovirus that can be used include type 1(e.g., strain Lang or T1L); type 2 (e.g., strain Jones or T2J); and type3 (e.g., strain Dearing or strain Abney, T3D or T3A); other strains ofreovirus can also be used. In a preferred embodiment, the reovirus ishuman reovirus serotype 3, more preferably the reovirus is humanreovirus serotype 3, strain Dearing. Alternatively, the reovirus can bea non-human mammalian reovirus (e.g., non-human primate reovirus, suchas baboon reovirus; equine; or canine reovirus), or a non-mammalianreovirus (e.g., avian reovirus). A combination of different serotypesand/or different strains of reovirus, such as reovirus from differentspecies of animal, can be used.

The reovirus may be naturally occurring or modified. The reovirus is“naturally-occurring”: when it can be isolated from a source in natureand has not been intentionally modified by humans in the laboratory. Forexample, the reovirus can be from a “field source”: that is, from ahuman patient.

The reovirus may be modified but still capable of lytically infecting amammalian cell having an active ras pathway. The reovirus may bechemically or biochemically pretreated (e.g., by treatment with aprotease, such as chymotrypsin or trypsin) prior to administration tothe proliferating cells. Pretreatment with a protease removes the outercoat or capsid of the virus and may increase the infectivity of thevirus. The reovirus may be coated in a liposome or micelle (Chandron andNibert, “Protease cleavage of reovirus capsid protein mu1 and mu1C isblocked by alkyl sulfate detergents, yielding a new type of infectioussubvirion particle”, J. of Virology 72(1):467-75 (1998)) to reduce orprevent an immune response from a mammal which has developed immunity tothe reovirus. For example, the virion may be treated with chymotrypsinin the presence of micelle forming concentrations of alkyl sulfatedetergents to generate a new infectious subvirion particle.

The reovirus may be a recombinant reovirus from two or more types ofreoviruses with differing pathogenic phenotypes such that it containsdifferent antigenic determinants thereby reducing or preventing animmune response by a mammal previously exposed to a reovirus subtype.Such recombinant virions can be generated by co-infection of mammaliancells with different subtypes of reovirus with the resulting resortingand incorporation of different subtype coat proteins into the resultingvirion capsids.

The reovirus may be modified by incorporation of mutated coat proteins,such as for example σ1, into the virion outer capsid. The proteins maybe mutated by replacement, insertion or deletion. Replacement includesthe insertion of different amino acids in place of the native aminoacids. Insertions include the insertion of additional amino acidresidues into the protein at one or more locations. Deletions includedeletions of one or more amino acid residues in the protein. Suchmutations may be generated by methods known in the art. For example,oligonucleotide site directed mutagenesis of the gene encoding for oneof the coat proteins could result in the generation of the desiredmutant coat protein. Expression of the mutated protein in reovirusinfected mammalian cells in vitro such as COS1 cells will result in theincorporation of the mutated protein into the reovirus virion particle(Turner and Duncan, “Site directed mutagenesis of the C-terminal portionof reovirus protein sigma1: evidence for a conformation-dependentreceptor binding domain” Virology 186(1):219-27 (1992); Duncan et al.,“Conformational and functional analysis of the C-terminal globular headof the reovirus cell attachment protein” Virology 182(2):810-9 (1991);Mah et al. “The N-terminal quarter of reovirus cell attachment proteinsigma 1 possesses intrinsic virion-anchoring function” Virology179(1):95-103 (1990))

The reovirus is preferably a reovirus modified to reduce or eliminate animmune reaction to the reovirus. Such modified reovirus are termed“immunoprotected reovirus”. Such modifications could include packagingof the reovirus in a liposome, a micelle or other vehicle to mask thereovirus from the mammals immune system. Alternatively, the outer capsidof the reovirus virion particle may be removed since the proteinspresent in the outer capsid are the major determinant of the hosthumoral and cellular responses.

A “proliferative disorder” is any cellular disorder in which the cellsproliferate more rapidly than normal tissue growth. Thus a“proliferating cell” is a cell that is proliferating more rapidly thannormal cells. The proliferative disorder, includes but is not limited toneoplasms. A neoplasm is an abnormal tissue growth, generally forming adistinct mass, that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoetic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. A tumor is the neoplastic growth of thedisease cancer. As used herein, a “neoplasm”, also referred to as a“tumor”, is intended to encompass hematopoietic neoplasms as well assolid neoplasms. Other proliferative disorders include, but are notlimited to neurofibromatosis.

At least some of the cells of the proliferative disorder have a mutationin which the Ras gene (or an element of the Ras signaling pathway) isactivated, either directly (e.g., by an activating mutation in Ras) orindirectly (e.g., by activation of an upstream element in the Raspathway). Activation of an upstream element in the Ras pathway includes,for example, transformation with epidermal growth factor receptor (EGFR)or Sos. A proliferative disorder that results, at least in part, by theactivation of Ras, an upstream element of Ras, or an element in the Rassignalling pathway is referred to herein as a “Ras-mediatedproliferative disorder”.

One neoplasm that is particularly susceptible to treatment by themethods of the invention is pancreatic cancer, because of the prevalenceof Ras-mediated neoplasms associated with pancreatic cancer. Otherneoplasms that are particularly susceptible to treatment by the methodsof the invention include breast cancer, central nervous system cancer(e.g., neuroblastoma and glioblastoma), peripheral nervous systemcancer, lung cancer, prostate cancer, colorectal cancer, thyroid cancer,renal cancer, adrenal cancer, liver cancer, lymphoma and leukemia. Oneproliferative disorder that is particularly susceptible to treatment bythe methods of this invention include neurofibromatosis 1 because of theactivation of the ras pathway.

“Administration to a proliferating cell or neoplasm” indicates that thereovirus is administered in a manner so that it contacts theproliferating cells or cells of the neoplasm (also referred to herein as“neoplastic cells”). The route by which the reovirus is administered, aswell as the formulation, carrier or vehicle, will depend on the locationas well as the type of the neoplasm. A wide variety of administrationroutes can be employed. For example, for a solid neoplasm that isaccessible, the reovirus can be administered by injection directly tothe neoplasm. For a hematopoietic neoplasm, for example, the reoviruscan be administered intravenously or intravascularly. For neoplasms thatare not easily accessible within the body, such as metastases or braintumors, the reovirus is administered in a manner such that it can betransported systemically through the body of the mammal and therebyreach the neoplasm (e.g., intrathecally, intravenously orintramuscularly). Alternatively, the reovirus can be administereddirectly to a single solid neoplasm, where it then is carriedsystemically through the body to metastases. The reovirus can also beadministered subcutaneously, intraperitoneally, topically (e.g., formelanoma), orally (e.g., for oral or esophageal neoplasm), rectally(e.g., for colorectal neoplasm), vaginally (e.g., for cervical orvaginal neoplasm), nasally or by inhalation spray (e.g., for lungneoplasm).

Reovirus can be administered systemically to mammals which are immunecompromised or which have not developed immunity to the reovirusepitopes. In such cases, reovirus administered systemically, i.e. byintraveneous injection, will contact the proliferating cells resultingin lysis of the cells.

Immunocompetent mammals previously exposed to a reovirus subtype mayhave developed humoral and/or cellular immunity to that reovirussubtype. Nevertheless, it has been found that direct injection of thereovirus into a solid tumor in immunocompetent mammals will result inthe lysis of the neoplastic cells.

On the other hand, when the reovirus is administered systemically toimmunocompetent mammals, the mammals may produce an immune response tothe reovirus. Such an immune response may be avoided if the reovirus isof a subtype to which the mammal has not developed immunity, or thereovirus has been modified as previously described herein such that itis immunoprotected, for example, by protease digestion of the outercapsid or packaging in a micelle.

Alternatively, it is contemplated that the immunocompetency of themammal against the reovirus may be suppressed either by theco-administration of pharmaceuticals known in the art to suppress theimmune system in general (Cuff et al., “Enteric reovirus infection as aprobe to study immunotoxicity of the gastrointestinal tract”Toxicological Sciences 42(2):99-108 (1998)) or alternatively theadministration of anti-antireovirus antibodies. The humoral immunity ofthe mammal against reovirus may also be temporarily reduced orsuppressed by plasmaphoresis of the mammals blood to remove theanti-reovirus antibodies. The humoral immunity of the mammal againstreovirus may additionally be temporarily reduced or suppressed by theintraveneous administration of non-specific immunoglobulin to themammal.

It is contemplated that the reovirus may be administered toimmunocompetent mammals immunized against the reovirus in conjunctionwith the administration of anti-antireovirus antibodies.“Anti-antireovirus antibodies” are antibodies directed againstanti-reovirus antibodies. Such antibodies can be made by methods knownin the art. See for example “Antibodies: A laboratory manual” E. Harlowand D. Lane, Cold Spring Harbor Laboratory (1988). Suchanti-antireovirus antibodies may be administered prior to, at the sametime or shortly after the administration of the reovirus. Preferably aneffective amount of the anti-antireovirus antibodies are administered insufficient time to reduce or eliminate an immune response by the mammalto the administered reovirus.

The term “substantial lysis” means at least 10% of the proliferatingcells are lysed, more preferably of at least 50% and most preferably ofat least 75% of the cells are lysed. The percentage of lysis can bedetermined for tumor cells by measuring the reduction in the size of thetumor in the mammal or the lysis of the tumor cells in vitro.

A “mammal suspected of having a proliferative disorder” means that themammal may have a proliferative disorder or tumor or has been diagnosedwith a proliferative disorder or tumor or has been previously diagnosedwith a proliferative disorder or tumor, the tumor or substantially allof the tumor has been surgically removed and the mammal is suspected ofharboring some residual tumor cells.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the reoviruses associated with“pharmaceutically acceptable carriers or excipients”. In making thecompositions of this invention, the active ingredient/reovirus isusually mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier which can be in the form of a capsule, sachet,paper or other container. When the pharmaceutically acceptable excipientserves as a diluent, it can be a solid, semi-solid, or liquid material,which acts as a vehicle, carrier or medium for the active ingredient.Thus, the compositions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining, for example, up to 10% by weight of the active compound,soft and hard gelatin capsules, suppositories, sterile injectablesolutions, and sterile packaged powders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

For preparing solid compositions such as tablets, the principal activeingredient/reovirus is mixed with a pharmaceutical excipient to form asolid preformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein. Preferably the compositions are administered by the oral ornasal respiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the reovirus of the present invention in controlled amounts.The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, for example, U.S.Pat. No. 5,023,252, herein incorporated by reference. Such patches maybe constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences.

The reovirus or the pharmaceutical composition comprising the reovirusmay be packaged into convenient kits providing the necessary materialspackaged into suitable containers. It is contemplated the kits may alsoinclude chemotherapeutic agents and/or anti-antireovirus antibody.

The reovirus is administered in an amount that is sufficient to treatthe proliferative disorder (e.g., an “effective amount”). Aproliferative disorder is “treated” when administration of reovirus tothe proliferating cells effects lysis of the proliferating cells. Thismay result in a reduction in size of the neoplasm, or in a completeelimination of the neoplasm. The reduction in size of the neoplasm, orelimination of the neoplasm, is generally caused by lysis of neoplasticcells (“oncolysis”) by the reovirus. Preferably the effective amount isthat amount able to inhibit tumor cell growth. Preferably the effectiveamount is from about 1.0 pfu/kg body weight to about 10¹⁵ pfu/kg bodyweight, more preferably from about 10² pfu/kg body weight to about 10¹³pfu/kg body weight. For example, for treatment of a human, approximately10² to 10¹⁷ plaque forming units (PFU) of reovirus can be used,depending on the type, size and number of tumors present. The effectiveamount will be determined on an individual basis and may be based, atleast in part, on consideration of the type of reovirus; the chosenroute of administration; the individual's size, age, gender; theseverity of the patient's symptoms; the size and other characteristicsof the neoplasm; and the like. The course of therapy may last fromseveral days to several months or until diminution of the disease isachieved.

The reovirus can be administered in a single dose, or multiple doses(i.e., more than one dose). The multiple doses can be administeredconcurrently, or consecutively (e.g., over a period of days or weeks).The reovirus can also be administered to more than one neoplasm in thesame individual.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 10² pfus to about 10¹³ pfus of thereovirus. The term “unit dosage forms” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of reovirus calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient.

It has been found that the reovirus is effective for the treatment ofsolid neoplasms in immunocompetent mammals. Administration of unmodifiedreovirus directly to the neoplasm results in oncolysis of the neoplasticcells and reduction in the size of the tumor.

It is contemplated that the reovirus may be administered in conjunctionwith surgery or removal of the neoplasm. Therefore, provided herewithare methods for the treatment of a solid neoplasm comprising surgicalremoval of the neoplasm and administration of a reovirus at or near tothe site of the neoplasm.

It is contemplated that the reovirus may be administered in conjunctionwith or in addition to radiation therapy.

It is further contemplated that the reovirus of the present inventionmay be administered in conjunction with or in addition to knownanticancer compounds or chemotherapeutic agents. Chemotherapeutic agentsare compounds which may inhibit the growth of tumors. Such agents,include, but are not limited to, 5-fluorouracil, mitomycin C,methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone,anthracyclins (Epirubicin and Doxurubicin), antibodies to receptors,such as herceptin, etopside, pregnasome, platinum compounds such ascarboplatin and cisplatin, taxanes such as taxol and taxotere, hormonetherapies such as tamoxifen and anti-estrogens, interferons, aromataseinhibitors, progestational agents and LHRH analogs.

Preferably the reovirus is administered in the absence of1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). For example, the1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is not administered to themammal either before, during or after the mammal receives the reovirus.

The reovirus of the present invention have been found to reduce thegrowth of tumors that are metastatic. In an embodiment of the invention,a method is provided for reducing the growth of metastatic tumors in amammal comprising administering an effective amount of a reovirus to themammal.

Utility

The reoviruses of the present invention may be used for a variety ofpurposes. They may be used in methods for treating ras-mediatedproliferative disorders in a mammal. The reovirus may be used to reduceor eliminate neoplasms. They may be used in methods for treatingmetastases. They may be used in conjunction with known treatments forcancer including surgery, chemotherapy and radiation.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given but are not meant tolimit the scope of the claims in any way.

EXAMPLES

In the examples below, all temperatures are in degrees Celsius (unlessotherwise indicated) and all percentages are weight percentages (alsounless otherwise indicated).

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning:

μM = micromolar mM = millimolar M = molar ml = milliliter μl =microliter mg = milligram μg = microgram PAGE = polyacrylamide gelelectrophoresis rpm = revolutions per minute FBS = fetal bovine serumDTT = dithiothrietol SDS = sodium dodecyl sulfate PBS = phosphatebuffered saline DMEM = Dulbecco's modified Eagle's medium α-MEM =α-modified Eagle's medium β-ME = β-mercaptoethanol MOI = multiplicity ofinfection PFU = plaque forming units MAPK = MAP kinase phosph-MAPK =phosphorylated-MAP kinase HRP = horseradish-peroxidase PKR =double-stranded RNA activated protein kinase RT-PCR = reversetranscriptase-polymerase chain reaction GAPDH =glyceraldehyde-3-phosphate dehydrogenase EGFR = epidermal growth factorreceptors MEK kinase = mitogen-activated extracellular signal-regulatedkinase DMSO = dimethylsulfoxide SCID = severe combined immunodeficiencyGeneral MethodsCells and Virus

Parental NIH-3T3 and NIH-3T3 cells transfected with the Harvey-ras(H-ras) and EJ-ras oncogenes were a generous gift of Dr. Douglas Faller(Boston University School of Medicine). NIH-3T3 cells along with theirSos-transformed counterparts (designated TNIH#5) were a generous gift ofDr. Michael Karin (University of California, San Diego). Dr. H.-J. Kung(Case Western Reserve University) kindly donated parental NIH-3T3 cellsalong with NIH-3T3 cells transfected with the v-erbB oncogene(designated THC-11). 2H1 cells, a derivative of the C3H 10T1/2 murinefibroblast line, containing the Harvey-ras gene under thetranscriptional control of the mouse metallothionein-I promoter wereobtained from Dr. Nobumichi Hozumi (Mount Sinai Hospital ResearchInstitute). These 2H1 cells are conditional ras transformant thatexpress the H-ras oncogene in the presence of 50 μM ZnSO₄. All celllines were grown in Dulbecco's modified Eagle's medium (DMEM) containing10% fetal bovine serum (FBS).

The NIH-3T3 tet-myc cells were obtained from Dr. R. N. Johnston(University of Calgary) and were grown in DMEM containing 10%heat-inactivated FBS and antibiotics in the presence or absence of 2μg/ml tetracycline (Helbing, C. C. et al., Cancer Res. 57:1255-1258(1997)). In the presence of tetracycline, expression of the human c-mycgene is repressed. Removal of tetracycline results in the elevation ofexpression of c-myc by up to 100-fold in these cells, which also displaya transformed phenotype.

The PKR⁺/⁻ and PKR^(o)/^(o) mouse embryo fibroblasts (MEFs) wereobtained from Dr. B. R. G. Williams (the Cleveland Clinic Foundation)and were grown in α-MEM containing fetal bovine serum and antibiotics aspreviously described (Yang, Y. L. et al. EMBO J. 14:6095-6106 (1995);Der, S. D. et al., Proc. Natl. Acad. Sci. USA 94:3279-3283 (1997)).

The Dearing strain of reovirus serotype 3 used in these studies waspropagated in suspension cultures of L cells and purified according toSmith (Smith, R. E. et al., (1969) Virology, 39:791-800) with theexception that β-mercaptoethanol (β-ME) was omitted from the extractionbuffer. Reovirus labelled with [³⁵S]methionine was grown and purified asdescribed by McRae and Joklik (McRae, M. A. and Joklik, W. K., (1978)Virology, 89:578-593). The particle/PFU ratio for purified reovirus wastypically 100/1.

Immunofluorescent Analysis of Reovirus Infection

For the immunofluorescent studies the NIH-3T3, TNIH#5, H-ras, EJ-ras,2H1 (+/−ZnSO₄), and THC-11 cells were grown on coverslips, and infectedwith reovirus at a multiplicity of infection (MOI) of ˜10 PFU cell ormock-infected by application of the carrier agent (phosphate-bufferedsaline, PBS) to the cells in an identical fashion as the administrationof virus to the cells. At 48 hours postinfection, cells were fixed in anethanol/acetic acid (20/1) mixture for 5 minutes, then rehydrated bysequential washes in 75%, 50% and 25% ethanol, followed by four washeswith phosphate-buffered saline (PBS). The fixed and rehydrated cellswere then exposed to the primary antibody (rabbit polyclonalanti-reovirus type 3 serum diluted 1/100 in PBS) [antiserum prepared byinjection of rabbits with reovirus serotype 3 in Freund's completeadjuvant, and subsequent bleedings] for 2 hours at room temperature.Following three washes with PBS, the cells were exposed to the secondaryantibody [goat anti-rabbit IgG (whole molecule)-fluoresceinisothiocyanate conjugate (FITC) [Sigma ImmunoChemicals F-0382] diluted1/100 in PBS containing 10% goat serum and 0.005% Evan's Blue] for 1hour at room temperature. Finally, the fixed and treated cells werewashed three more times with PBS and then once with double-distilledwater, dried and mounted on slides in 90% glycerol containing 0.1%phenylenediamine, and viewed with a Zeiss Axiophot microscope on whichCarl Zeiss camera was mounted (the magnification for all pictures was200×).

Detection of MAP Kinase (ERK) Activity

The PhosphoPlus p44/42 MAP kinase (Thr202/Tyr204) Antibody kit (NewEngland Biolabs) was used for the detection of MAP kinase in celllysates according to the manufacturer's instructions. Briefly,subconfluent monolayer cultures were lysed with the recommendedSDS-containing sample buffer, and subjected to SDS-PAGE, followed byelectroblotting onto nitrocellulose paper. The membrane was then probedwith the primary antibody (anti-total MAPK or anti-phospho-MAPK),followed by the horseradish peroxidase (HRP)-conjugated secondaryantibody as described in the manufacturer's instruction manual.

Radiolabelling of Reovirus-Infected Cells and Preparation of Lysates

Confluent monolayers of NIH-3T3, TNIH#5, H-ras, EJ-ras, 2H1 (+/−ZnSO₄),and THC-11 cells were infected with reovirus (MOI ˜10 PFU/cell). At 12hours postinfection, the media was replaced with methionine-free DMEMcontaining 10% dialyzed FBS and 0.1 mCi/ml [³⁵S]methionine. Afterfurther incubation for 36 hours at 37° C., the cells were washed inphosphate-buffered saline (PBS) and lysed in the same buffer containing1% Triton X-100, 0.5% sodium deoxycholate and 1 mM EDTA. The nuclei werethen removed by low speed centrifugation and the supernatants werestored at −70° C. until use.

Preparation of Cytoplasmic Extracts for In Vitro Kinase Assays

Confluent monolayers of the various cell lines were grown on 96 wellcell culture plates. At the appropriate time postinfection the media wasaspirated off and the cells were lysed with a buffer containing 20 mMHEPES [pH 7.4], 120 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.5%Nonidet P40, 2 μg/ml leupeptin, and 50 μg/ml aprotinin. The nuclei werethen removed by low-speed centrifugation and the supernatants werestored at −70° C. until use.

Cytoplasmic extracts were normalized for protein concentrations beforeuse by the Bio-Rad protein microassay method. Each in vitro kinasereaction contained 20 μl of cell extract, 7.5 μl of reaction buffer (20mM HEPES [pH 7.4], 120 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, and 10%glycerol) and 7.0 μl of ATP mixture (1.0 μCi[γ-³²P] ATP in 7 μl ofreaction buffer), and was incubated for 30 minutes at 37° C. (Mundschau,L. J., and Faller, D. V., J. Biol. Chem., 267:23092-23098 (1992)).Immediately after incubation the labelled extracts were either boiled inLaemmli SDS-sample buffer or were either precipitated withagarose-poly(I)poly(C) beads or immunoprecipitated with an anti-PKRantibody.

Agarose Poly(I)Poly(C) Precipitation

To each in vitro kinase reaction mixture, 30 μl of a 50% Agpoly(I)poly(C) Type 6 slurry (Pharmacia LKB Biotechnology) was added,and the mixture was incubated at 4° C. for 1 h. The Ag poly(I)poly(C)beads with the absorbed, labelled proteins were then washed four timeswith wash buffer (20 mM HEPES [7.5 pH], 90 mM KCl, 0.1 mM EDTA, 2 mMdithiothreitol, 10% glycerol) at room temperature and mixed with 2×Laemmli SDS sample buffer. The beads were then boiled for 5 min, and thereleased proteins were analyzed by SDS-PAGE.

Polymerase Chain Reaction

Cells at various times postinfection were harvested and resuspended inice cold TNE (10 mM Tris [pH 7.8], 150 mM NaCl, 1 mM EDTA) to whichNP-40 was then added to a final concentration of 1%. After 5 minutes,the nuclei were pelleted and RNA was extracted from the supernatantusing the phenol:chloroform procedure. Equal amounts of total cellularRNA from each sample were then subjected to RT-PCR (Wong, H. et al.,(1994) Anal. Biochem. 223:251-258) using random hexanucleotide primers(Pharmacia) and RTase (GIBCO-BRL) according to the manufacturers'protocol. The cDNAs from the RT-PCR step were then subjected toselective amplification of reovirus cDNA using appropriate primers thatamplify a predicted 116 bp fragment. These primer sequences were derivedfrom the S1 sequence determined previously (Nagata, L. et al., (1984)Nucleic Acids Res. 12:8699-8710). The GAPDH primers of Wong, H. et al.,(1994) Anal. Biochem. 223:251-258 were used to amplify a predicted 306bp GAPDH fragment which served as a PCR and gel loading control.Selective amplification of the s1 and GAPDH cDNA's was performed usingTaq DNA polymerase (GIBCO-BRL) according to the manufacturers' protocolusing a Perkin Elmer Gene Amp PCR system 9600. PCR was carried out for28 cycles with each consisting of a denaturing step for 30 seconds at97° C., annealing step for 45 seconds at 55° C., and polymerization stepfor 60 seconds at 72° C. PCR products were analyzed by electrophoresisthrough an ethidium bromide-impregnated TAE-2% agarose gel andphotographed under ultra-violet illumination with Polaroid 57 film.

Immunoprecipitation and SDS-PAGE Analysis

Immunoprecipitation of ³⁵S-labelled reovirus-infected cell lysates withanti-reovirus serotype 3 serum was carried out as previously described(Lee, P. W. K. et al. (1981) Virology, 108:134-146). Immunoprecipitationof ³²P-labelled cell lysates with an anti-PKR antibody (from Dr. MichaelMathews, Cold Spring Harbor) was similarly carried out.Immunoprecipitates were analyzed by discontinuous SDS-PAGE according tothe protocol of Laemmli (Laemmli, U. K., (1970) Nature, 227:680-685).

Example 1 Activated Intermediates in the Ras Signalling Pathway AugmentReovirus Infection Efficiency

It was previously shown that 3T3 cells and their derivatives lackingepidermal growth factor receptors (EGFR) are poorly infectible byreovirus, whereas the same cells transformed with either EGFR or v-erb Bare highly susceptible as determined by cytopathic effects, viralprotein synthesis, and virus output (Strong, J. E. et al., (1993)Virology, 197:405-411; Strong, J. E. and Lee, P. W. K., (1996) J.Virol., 70:612-616).

To determine whether downstream mediators of the EGFR signaltransduction pathway may be involved, a number of different NIH3T3-derived, transformed with constitutively activated oncogenesdownstream of the EGFR, were assayed for relative susceptibility toreovirus infection. Of particular interest were intermediates in the rassignalling pathway (reviewed by Barbacid, M., Annu. Rev. Biochem.,56:779-827 (1987); Cahill, M. A., et al., Curr. Biol., 6:16-19 (1996).To investigate the Ras signalling pathway, NIH 3T3 parental cell linesand NIH 3T3 lines transfected with activated versions of Sos (Aronheim,A., et al., (1994) Cell, 78:949-961) or ras (Mundschau, L. J. andFaller, D. V., (1992) J. Biol. Chem., 267:23092-23098) oncogenes wereexposed to reovirus, and their capacity to promote viral proteinsynthesis was compared.

Detection of viral proteins was initially carried out using indirectimmunofluorescent microscopy as described above. The results indicatedthat whereas the NIH 3T3 cells adopted a typically flattened, spread-outmorphology with marked contact inhibition, the transformed cells allgrew as spindle-shaped cells with much less contact inhibition. Oncomparing the uninfected parental cell lines with the varioustransformed cell lines, it was apparent that the morphology of the cellswas quite distinct upon transformation. Upon challenge with reovirus, itbecame clear that parental NIH 3T3 line was poorly infectible (<5%),regardless of the source of the parental NIH 3T3 line. In contrast, thetransfected cell lines each demonstrated relatively pronouncedimmunofluorescence by 48 hours postinfection (data not shown).

To demonstrate that viral protein synthesis was more efficient in theSos- or Ras-transformed cell lines, cells were continuously labeled with[³⁵S]-methionine from 12 to 48 hr postinfection and the proteins wereanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), as described above.

The results showed clearly that the levels of viral protein synthesiswere significantly higher in the Sos- or Ras-transformed cells than inparental NIH 3T3 cells. The identities of the viral bands were confirmedby immunoprecipitation of the labeled proteins with polyclonalanti-reovirus antibodies. Since the uninfected NIH 3T3 cells and theirtransformed counterparts displayed comparable levels of cellular proteinsynthesis and doubling times (data not shown), the observed differencein the level of viral protein synthesis could not be due to intrinsicdifferences in growth rates or translation efficiencies for these celllines.

The long-term fate of infected NIH-3T3 cells was followed by passagingthese cells for at least 4 weeks. They grew normally and appearedhealthy, with no sign of lytic or persistent infection; no virus couldbe detected in the medium after this time (data not shown).

Example 2 Enhanced Infectibility Conferred By Activated Oncogenes is notDue to Long-Term Transformation or the Generalized Transformed State ofthe Cell

To determine whether the differences in susceptibility may be the resultof long-term effects of transformation, or the result of the activatedoncogene itself, a cell line expressing a zinc-inducible cellularHarvey-ras (c-H-ras) gene was tested for susceptibility to reovirusinfectibility, as described above. These cells, called 2H1, were derivedfrom the C3H 10T½ cell line which is poorly infectible by reovirus (datanot shown), and carry the c-H-ras gene under the control of the mousemetallothionine-I promoter (Trimble, W. S. et al. (1986) Nature,321:782-784).

Cells were either mock-treated or pretreated with 50 μM ZnSO₄ 18 hoursprior to infection or mock-infection (administration of carrier agent),followed by indirect immunofluorescent analysis of these cells at 48hours postinfection or mock-infection.

The results (not shown) demonstrated that uninduced cells were poorlyinfectible (<5%) whereas those induced for only 18 hours were much moresusceptible (>40%). Enhanced viral protein synthesis in the Zn-induced2H1 cells was further confirmed by metabolic labeling of the cells with[³⁵S]methionine followed by SDS-PAGE analysis of virus-specific proteins(not shown).

Based on these observations, the augmentation of reovirus infectionefficiency in the transformed cells is a direct result of the activatedoncogene product(s), and not due to other factors such as aneuploidyoften associated with long-term transformation, or other accumulatedmutations that may be acquired under a chronically transformed state(e.g., p53 or myc activation).

To show further that susceptibility to reovirus infection is not aresult of transformation per se (i.e., a result of the transformed stateof the host cell), NIH-3T3 cells containing a tetracycline-controlledhuman c-myc gene (tet-myc cells) were examined (Helbing, C. C. et al,Cancer Res. 57:1255-1258 (1997)). These cells normally are maintained intetracycline (2 μg/ml) which represses the expression of c-myc. Removalof tetracycline under normal growth conditions (10% fetal bovine serum)leads to accumulation of the c-Myc protein and the cells display atransformed phenotype. We found that these cells were unable to supportvirus growth either in the presence or in the absence of tetracycline(data not shown), suggesting that susceptibility to reovirus infectionis not due to the general transformed state of the host cell, but ratherrequires specific transformation by elements of the Ras signalingpathway.

A good indicator of activation of the Ras signaling pathway is theactivation of the MAP kinases ERK1 and ERK2 (for a review, see Robinson,M. J. and Cobb, M. H., Curr. Opin. Cell. Biol. 9:180-186 (1997)). Inthis regard, it was found that, compared with untransformed cells,Ras-transformed cells have a significantly higher ERK1/2 activity (datanot shown). Furthermore, an examination of a number of human cancer celllines has revealed an excellent correlation between the level of ERK1/2activity and susceptibility to reovirus infection (data not shown),although ERK1/2 itself does not appear to play any role in it. Mouse Lcells and human HeLa cells, in which reovirus grows very well, bothmanifest high ERK 1/2 activity (data not shown).

Example 3 Viral Transcripts are Generated but not Translated inReovirus-Resistant NIH 3T3 Cells

The step at which reovirus infection is blocked in nonsusceptible NIH3T3 cells was also identified. Because virus binding and virusinternalization for nonsusceptible cells were comparable to thoseobserved for susceptible cells (Strong, J. E. et al., (1993) Virology,197:405-411), the transcription of viral genes was investigated.

The relative amounts of reovirus S1 transcripts generated in NIH 3T3cells and the Ras-transformed cells during the first 12 hours ofinfection were compared after amplification of these transcripts bypolymerase chain reaction (PCR), as described above. The resultsdemonstrated that the rates of accumulation of S1 transcripts in the twocell lines were similar, at least up to 12 hours postinfection. Similardata were obtained when rates of accumulation of other reovirustranscripts were compared (data not shown). These results demonstratethat infection block in nonsusceptible cells is not at the level oftranscription of viral genes, but rather, at the level of translation ofthe transcripts.

At later times, the level of viral transcripts present in untransformedNIH-3T3 cells decreased significantly whereas transcripts in transformedcells continued to accumulate (data not shown). The inability of thesetranscripts to be translated in NIH-3T3 cells probably contributed totheir degradation. As expected, the level of viral transcripts ininfected L cells was at least comparable with that in infectedRas-transformed cells (data not shown).

Example 4 A 65 kDa Protein is Phosphorylated in Reovirus-Treated NIH 3T3Cells but not in Reovirus-Infected Transformed Cells

Because viral transcripts were generated, but not translated, in NIH 3T3cells, it was investigated whether the double-stranded RNA(dsRNA)-activated protein kinase, PKR, is activated (phosphorylated) inthese cells (for example, by S1 mRNA transcripts which have been shownto be potent activators of PKR (Bischoff, J. R. and Samuel, C. E.,(1989) Virology, 172:106-115), which in turn leads to inhibition oftranslation of viral genes. The corollary of such a scenario would bethat in the case of the transformed cells, this activation is prevented,allowing viral protein synthesis to ensue.

NIH 3T3 cells and v-erbB- or Ras-transformed cells (designated THC-11and H-ras, respectively) were treated with reovirus (i.e., infected) ormock-infected (as above), and at 48 hours post treatment, subjected toin vitro kinase reactions, followed by autoradiographic analysis asdescribed above.

The results clearly demonstrated that there was a distinctphosphoprotein migrating at approximately 65 kDa, the expected size ofPKR, only in the NIH 3T3 cells and only after exposure to reovirus. Thisprotein was not labeled in the lysates of either the uninfectedtransformed cell lines or the infected transformed cell lines. Instead,a protein migrating at approximately 100 kDa was found to be labeled inthe transformed cell lines after viral infection. This protein wasabsent in either the preinfection or the postinfection lysates of theNIH 3T3 cell line, and was not a reovirus protein because it did notreact with an anti-reovirus serum that precipitated all reovirusproteins (data not shown). A similar 100 kDa protein was also found tobe ³²P-labeled in in vitro kinase reactions of postinfection lysates ofthe Sos-transformed cell lines (data not shown).

That intermediates in the Ras signalling pathway were responsible forthe lack of phosphorylation of the 65 kDa protein was further confirmedby the use of the 2H1 cells which contain a Zn-inducible Ras oncogene.Uninduced 2H1 cells (relatively resistant to reovirus infection, asshown above), were capable of producing the 65 kDa phosphoprotein onlyafter exposure to virus. However, 2H1 cells subjected to Zn-induction ofthe H-Ras oncogene showed significant impairment of the production ofthis phosphoprotein. This impairment coincided with the enhancement ofviral synthesis. These results therefore eliminated the possibility thatthe induction of the 65 kDa phosphoprotein was an NIH 3T3-specificevent, and clearly established the role of Ras in preventing (orreversing) induction of the production of this phosphoprotein. TheZn-induced 2H1 cells did not produce the 100 kDa phosphoprotein seen inthe infected, chronically transformed H-Ras cells.

Example 5 Induction of Phosphorylation of the 65 kDa Protein RequiresActive Viral Transcription

Since production of the 65 kDa phosphoprotein occurred only in cellsthat were resistant to reovirus infection, and only after the cells wereexposed to reovirus, it was investigated whether active viraltranscription was required for production of the 68 kDa phosphoprotein.Reovirus was UV-treated to inactivate its genome prior to administrationof the reovirus to NIH 3T3 cells. For UV-treatment, reovirus wassuspended in DMEM to a concentration of approximately 4×10⁸ PFU/mL andexposed to short-wave (254 nm) UV light for 20 minutes. UV-inactivatedvirus were non-infectious as determined by lack of cytopathic effects onmouse L-929 fibroblasts and lack of viral protein synthesis by methodsof [³⁵S]-methionine labelling as previously described. Such UV treatmentabolished viral gene transcription, as analyzed by PCR, and hence viralinfectivity (data not shown). The cells were then incubated for 48hours, and lysates were prepared and subjected to in vitro ³²P-labelingas before. The results showed that NIH 3T3 cells infected with untreatedreovirus produced a prominent 65 kDa ³²P-labelled band not found inuninfected cells. Cells exposed to UV-treated reovirus behaved similarlyto the uninfected control cells, manifesting little phosphorylation ofthe 65 kDa protein. Thus, induction of the phosphorylation of the 65 kDaphosphoprotein is not due to dsRNA present in the input reovirus;rather, it requires de novo transcription of the viral genes, consistentwith the identification of the 65 kDa phosphoprotein as PKR.

Example 6 Identification of the 65 kDa Phosphoprotein as PKR

To determine whether the 65 kDa phosphoprotein was PKR, a dsRNA bindingexperiment was carried out in which poly(I)-poly(c) agarose beads wereadded to ³²P-labeled lysates, as described above. After incubation for30 minutes at 4° C., the beads were washed, and bound proteins werereleased and analyzed by SDS-PAGE. The results showed that the 65 kDaphosphoprotein produced in the postinfection NIH 3T3 cell lysates wascapable of binding to dsRNA; such binding is a well-recognizedcharacteristic of PKR. In contrast, the 100 kDa phosphoprotein detectedin the infected H-ras-transformed cell line did not bind to thePoly(I)-poly(c) agarose. The 65 kDa phosphoprotein was alsoimmunoprecipitable with a PKR-specific antibody (provided by Dr. MikeMathews, Cold Spring Harbor Laboratory), confirming that it was indeedPKR.

Example 7 PKR Inactivation or Deletion Results in Enhanced Infectibilityof Untransformed Cells

If PKR phosphorylation is responsible for the shut-off of viral genetranslation in NIH-3T3 cells, and one of the functions of the activatedoncogene product(s) in the transformed cells is the prevention of thisphosphorylation event, then inhibition of PKR phosphorylation in NIH-3T3cells by other means (e.g. drugs) should result in the enhancement ofviral protein synthesis, and hence infection, in these cells. To testthis idea, 2-aminopurine was used. This drug has been shown to possessrelatively specific inhibitory activity towards PKR autophosphorylation(Samuel, C. E. and Brody, M., (1990) Virology, 176:106-113; Hu, Y. andConway, T. W. (1993), J. Interferon Res., 13:323-328). Accordingly, NIH3T3 cells were exposed to 5 mM 2-aminopurine concurrently with exposureto reovirus. The cells were labeled with [³⁵S]methionine from 12 to 48 hpostinfection, and lysates were harvested and analyzed by SDS-PAGE.

The results demonstrated that exposure to 2-aminopurine resulted in asignificantly higher level of viral protein synthesis in NIH 3T3 cells(not shown). The enhancement was particularly pronounced afterimmunoprecipitating the lysates with an anti-reovirus serum. Theseresults demonstrate that PKR phosphorylation leads to inhibition ofviral gene translation, and that inhibition of this phosphorylationevent releases the translation block. Therefore, intermediates in theRas signalling pathway negatively regulate PKR, leading to enhancedinfectibility of Ras-transformed cells.

Interferon β, known to induce PKR expression, was found to significantlyreduce reovirus replication in Ras-transformed cells (data not shown).

A more direct approach to defining the role of PKR in reovirus infectionis through the use of cells that are devoid of PKR. Accordingly, primaryembryo fibroblasts from wild-type PKR⁺/⁺ and PKR^(o)/^(o) mice (Yang, Y.L. et al. EMBO J. 14:6095-6106 (1995)) were compared in terms ofsusceptibility to reovirus infection. The results clearly showed thatreovirus proteins were synthesized at a significantly higher level inthe PKR^(o)/^(o) cells than in the PKR⁺/⁺ cells. These experimentsdemonstrated that PKR inactivation or deletion enhanced host cellsusceptibility to reovirus infection in the same way as doestransformation by Ras or elements of the Ras signaling pathway, therebyproviding strong support of the role of elements of the Ras signalingpathway in negatively regulating PKR.

Example 8 Inactivation of PKR in Transformed Cells does not Involve MEK

Receptor tyrosine kinases such as EGFRs are known to stimulate themitogen-activated or extracellular signal-regulated kinases (ERK1/2) viaRas (see Robinson, M. J. and Cobb, M. H., Curr. Opin. Cell. Biol.9:180-186 (1997)). This stimulation requires the phosphorylation ofERK1/2 by the mitogen-activated extracellular signal-regulated kinase,kinase MEK, which itself is activated (phosphorylated) by Raf, aserine-threonine kinase downstream of Ras. To determine if MEK activitywas required for PKR inactivation in transformed cells, the effect ofthe recently identified MEK inhibitor PD98059 (Dudley, D. T. et al.,Proc. Natl. Acad. Sci. USA 92:7686-7689 (1995); Waters, S. D. et al., J.Biol. Chem. 270:20883-20886 (1995)) on infected Ras-transformed cellswas studied.

H-Ras-transformed cells were grown to 80% confluency and infected withreovirus at an MOI of approximately 10 p.f.u./cell. PD98059(Calbiochem), dissolved in dimethylsulfoxide (DMSO), was applied to thecells at the same time as the virus (final concentration of PD98059 was50 μM). The control cells received an equivalent volume of DMSO. Thecells were labeled with ³⁵S-methionine from 12 to 48 hourspost-infection. Lysates were then prepared, immunoprecipitated with thepolyclonal anti-reovirus serotype 3 serum and analyzed by SDS-PAGE.

The results (data not shown) showed that PD98059, at a concentrationthat effectively inhibited ERK1/2 phosphorylation, did not inhibitreovirus protein synthesis in the transformed cells. On the contrary,PD98059 treatment consistently caused a slight enhancement of viralprotein synthesis in these cells; the reason for this is underinvestigation. Consistent with the lack of inhibition of viral proteinsynthesis in the presence of PD98059, the PKR in these cells remainedunphosphorylated (data not shown). As expected, PD98059 had no effect onreovirus-induced PKR phosphorylation in untransformed NIH-3T3 cells(data not shown). These results indicated that MEK and ERK1/2 are notinvolved in PKR activation.

Example 9 In Vivo Oncolytic Capability of Reovirus

A severe combined immunodeficiency (SCID) host tumor model was used toassess the efficacy of utilizing reovirus for tumor reduction. Male andfemale SCID mice (Charles River, Canada) were injected withv-erbB-transformed NIH 3T3 mouse fibroblasts (designated THC-11 cells)in two subcutaneous sites overlying the hind flanks. In a first trial,an injection bolus of 2.3×10⁵ cells in 100 μl of sterile PBS was used.In a second trial, an injection bolus of 4.8×10⁶ cells in 100 μl PBS wasused. Palpable tumors were evident approximately two to three weeks postinjection.

Reovirus serotype three (strain Dearing) was injected into theright-side tumor mass (the “treated tumor mass”) in a volume of 20 μl ata concentration of 1.0×10⁷ plaque forming units (PFU)/ml. The left-sidetumor mass (the “untreated tumor mass”) was left untreated. The micewere observed for a period of seven days following injection withreovirus, measurements of tumor size were taken every two days usingcalipers, and weight of tumors was measured after sacrifice of theanimals. All mice were sacrificed on the seventh day. Results are shownin Table 1.

TABLE 1 Tumor Mass after Treatment with Reovirus Trial 1 (n = 8) meanuntreated tumor mass 602 mg mean treated tumor mass 284 mg Trial 2 (n =12) mean control tumor mass 1523.5 mg mean untreated tumor mass 720.9 mgmean treated tumor mass 228.0 mgThe treated tumor mass was 47% of that of the untreated tumor mass intrial 1, and 31.6% of the untreated tumor mass in trial 2. These resultsindicated that the virus-treated tumors were substantially smaller thanthe untreated tumors, and that there may be an additional systemiceffect of the virus on the untreated tumor mass.

Similar experiments were also conducted using unilateral introduction oftumor cells. SCID mice were injected subcutaneously and unilaterally inthe hind flank with v-erbB-transformed NIH 3T3 mouse fibroblasts (THC-11cells). Palpable tumors (mean area 0.31 cm²) were established after twoweeks. Eight animals were then given a single intratumoral injection of1.0×10⁷ PFUs of reovirus serotype 3 (strain Dearing) inphosphate-buffered saline (PBD). Control tumors (n=10) were injectedwith equivalent amounts of UV-inactivated virus. Tumor growth wasfollowed for 12 days, during which time no additional reovirus treatmentwas administered.

Results, shown in FIG. 2, demonstrated that treatment of these tumorswith a single dose of active reovirus (open circles) resulted indramatic repression of tumor growth by the thirteenth day (endpoint),when tumors in the control animals injected with a single dose ofinactivated reovirus (closed circles) exceeded the acceptable tumorburden. This experiment was repeated several times and found to behighly reproducible, thus further demonstrating the efficacy of reovirusin repressing tumor growth.

Example 10 In Vivo Oncolytic Capability of Reovirus Against Human BreastCancer-Derived Cell Lines

In vivo studies were also carried out using human breast carcinoma cellsin a SCID mouse model. Female SCID mice were injected with 1×10⁶ humanbreast carcinoma MDA-MB468 cells in two subcutaneous sites, overlyingboth hind flanks. Palpable tumors were evident approximately two to fourweeks post injection. Undiluted reovirus serotype three (strain Dearing)was injected into the right side tumor mass in a volume of 20 μl at aconcentration of 1.0×10⁷ PFU/ml. The following results were obtained:

TABLE 2 Tumor Mass After Treatment with Reovirus mean untreated tumormean treated tumor mass TREATMENT mass (left side) (right side) Reovirus(N = 8) 29.02 g 38.33 g Control (N = 8) 171.8 g 128.54 g *Note: One ofthe control mice died early during the treatment phase. None of thereovirus-treated mice died.

Although these studies were preliminary, it was clear that the size ofthe rumors in the reovirus-treated animals was substantially lower thanthat in the untreated animals. However, the size of the tumors on theright (treated) side of the reovirus-treated animals was slightly largeron average than the left (untreated) side. This was unexpected but maybe explained by the composition of the mass being taken up byinflammatory cells with subsequent fibrosis, as well as by the fact thatthese tumors were originally larger on the right side on average thanthe left. The histologic composition of the tumor masses is beinginvestigated. These results also support the systemic effect thereovirus has on the size of the untreated tumor on the contralateralslide of reovirus injection.

Example 11 Susceptibility of Additional Human Tumors to ReovirusOncolysis

In view of the in vivo results presented above, the oncolytic capabilityobserved in murine cells was investigated in cell lines derived fromadditional human tumors.

Cells and Virus

All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum (FBS).

The Dearing strain of reovirus serotype 3 used in these studies waspropagated in suspension cultures of L cells and purified according toSmith (Smith, R. E. et al., (1969) Virology, 39:791-800) with theexception that β-mercaptoethanol (β-ME) was omitted from the extractionbuffer. Reovirus labelled with [³⁵S]methionine was grown and purified asdescribed by McRae and Joklik (McRae, M. A. and Joklik, W. K, (1978)Virology, 89:578-593). The particle/PFU ration for purified reovirus wastypically 100/1.

Cytopathic Effects of Reovirus on Cells

Confluent monolayers of cells were infected with reovirus serotype 3(strain Dearing) at a multiplicity of infection (MOI) of approximately40 plaque forming units (PFU) per cell. Pictures were taken at 36 hourpostinfection for both reovirus-infected and mock-infected cells.

Immunofluorescent Analysis of Reovirus Infection

For the immunofluorescent studies the cells were grown on coverslips,and infected with reovirus at a multiplicity of infection (MOI) of ˜10PFU/cell or mock-infected as described above. At various timespostinfection, cells were fixed in an ethanol/acetic acid (20/1) mixturefor 5 minutes, then rehydrated by subsequential washes in 75%, 50% and25% ethanol, followed by 4 washes with phosphate-buffered saline (PBS).The fixed and rehydrated cells were then exposed to the primary antibody(rabbit polyclonal anti-reovirus type 3 serum diluted 1/100 in PBS) for2 hr at room temperature. Following 3 washes with PBS, the cells wereexposed to the secondary antibody [goat anti-rabbit IgG (whole molecule)fluorescein isothiocyanate (FITC) conjugate diluted 1/100 in PBScontaining 10% goat serum and 0.005% Evan's Blue counterstain] for 1hour at room temperature. Finally, the fixed and treated cells werewashed 3 more times with PBS, followed by 1 wash with double-distilledwater, dried and mounted on slides in 90% glycerol containing 0.1%phenylenediamine, and viewed with a Zeiss Axiophot microscope mountedwith a Carl Zeiss camera (magnification for all pictures was 200×).

Infection of Cells and Quantitation of Virus

Confluent monolayers of cells grown in 24-well plates were infected withreovirus at an estimated multiplicity of 10 PFU/cell. After 1 hourincubation at 37° C., the monolayers were washed with warm DMEM-10% FBS,and then incubated in the same medium. At various times postinfection, amixture of NP-40 and sodium deoxycholate was added directly to themedium on the infected monolayers to final concentrations of 1% and0.5%, respectively. The lysates were then harvested and virus yieldswere determined by plaque titration on L-929 cells.

Radiolabelling Of Reovirus-Infected Cells and Preparation of Lysates

Confluent monolayers of cells were infected with reovirus (MOI ˜10PFU/cell). At various times postinfection, the media was replaced withmethionine-free DMEM containing 10% dialyzed PBS and 0.1 mCi/ml[³⁵S]methionine. After further incubation for 1 hour at 37° C., thecells were washed in phosphate-buffered saline (PBS) and lysed in thesame buffer containing 1% Triton X-100, 0.5% sodium deoxycholate and 1mM EDTA. The nuclei were then removed by low speed centrifugation andthe supernatants was stored at 70° C. until use.

Immunoprecipitation and SDS-PAGE Analysis

Immunoprecipitation of [³⁵S]-labelled reovirus-infected cell lysateswith anti-reovirus serotype 3 serum was carried out as previouslydescribed (Lee, P. W. K. et al. (1981) Virology, 108:134-146).Immunoprecipitates were analyzed by discontinuous SDS-PAGE according tothe protocol of Laemmli (Laemmli, U. K., (1970) Nature, 227:680-685).

Breast Cancer

The c-erbB-2/neu gene encodes a transmembrane protein with extensivehomology to the EGFR that is overexpressed in 20-30% of patients withbreast cancer (Yu, D. et al. (1996) Oncogene 13:1359). Since it has beenestablished herein that Ras activation, either through point mutationsor through augmented signaling cascade elements upstream of Ras(including the c-erbB-2/neu homologue EGFR) ultimately creates ahospitable environment for reovirus replication, an array of cell linesderived from human breast cancers were assayed for reovirussusceptibility. The cell lines included MDA-MD-435SD (ATCC depositHTB-129), MCF-7 (ATCC deposit HTB-22), T-27-D (ATCC deposit HTB-133),BT-20 (ATCC deposit HTB-19), HBL-100 (ATCC deposit HTB-124), MDA-MB-468(ATCC deposit HTB-132), and SKBR-3 (ATCC deposit HTB-30).

Based upon induction of cytopathic effects, and viral protein synthesisas measured by radioactive metabolic labeling and immunofluorescence asdescribed above, it was found that five out of seven of the testedbreast cancers were susceptible to reovirus infection: MDA-MB-435S,MCF-7, T-27-D, MDA MB-468, and SKBR-3 were exquisitely sensitive toinfection, while BT-20 and HBL-100 demonstrated no infectibility.

Brain Glioblastoma

Next a variety of cell lines derived from human brain glioblastomas wasinvestigated. The cell lines included A-172, U-118, U-178, U-563, U-251,U-87 and U-373 (cells were a generous gift from Dr. Wee Yong, Universityof Calgary).

Six out of seven glioblastoma cell lines demonstrated susceptibility toreovirus infection, including U-118, U-178, U-563, U-251, U-87 andU-373, while A-172 did not demonstrate any infectibility, as measured bycytopathic effects, immunofluorescence and [³⁵S]-methionine labeling ofreovirus proteins.

The U-87 glioblastoma cell line was investigated further. To assess thesensitivity of U-87 cells to reovirus, U-87 cells (obtained from Dr. WeeYong, University of Calgary) were grown to 80% confluency and were thenchallenged with reovirus at a multiplicity of infection (MOI) of 10.Within a period of 48 hours there was a dramatic, widespread cytopathiceffect (data not shown). To demonstrate further that the lysis of thesecells was due to replication of reovirus, the cells were thenpulse-labeled with [³⁵S]methionine for three hour periods at varioustimes postinfection and proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as describedabove. The results (not shown) clearly demonstrated effective reovirusreplication within these cells with resultant shutoff of host proteinsynthesis by 24 hours postinfection.

The U-87 cells were also introduced as human tumor xenografts into thehind flank of 10 SCID mice. U-87 cells were grown in Dulbecco's modifiedEagle's medium containing 10% fetal bovine serum, as described above.Cells were harvested, washed, and resuspended in sterile PBS; 2.0×10⁶cells in 100 μl were injected subcutaneously at a site overlying thehind flank in five- to eight-week old male SCID mice (Charles River,Canada). Tumor growth was measured twice weekly for a period of fourweeks. Results, shown in FIG. 3, demonstrated that treatment of U-87tumors with a single intratumoral injection of 1.0×10⁷ PFUs of livereovirus (open circles, n=5) resulted in drastic repression of tumorgrowth, including tumor regression by the fourth week post-treatment(P=0.008), in comparison with treatment of tumors with a singleintratumoral injection of the same amount of UV-inactivated reovirus(closed circles, n=5).

Hematoxylin/eosin (HE)-staining of the remaining microfoci of the tumorstreated with active virus, performed as described (H. Lyon, CellBiology, A Laboratory Handbook, J. E. Celis, ed., Academic Press, 1994,p. 232) revealed that the remaining tumor mass consisted largely ofnormal stroma without appreciable numbers of viable tumor cells, nor wasthere any evidence of infiltration of rumor cells into the underlyingskeletal muscle (data not shown). Necrosis of tumor cells was due todirect lysis by the virus, the same mechanism of cell killing as byreovirus in vitro.

To determine if there was viral spread beyond the tumor mass,immunofluorescent microscopy using antibodies directed against totalreovirus proteins was conducted as described above, on paraffin sectionsof the tumor and adjoining tissue. It was found that reovirus-specificproteins were confined to the tumor mass; no viral staining was detectedin the underlying skeletal muscle (data not shown). As expected, viralproteins were not present in tumors injected with the UV-inactivatedvirus (data not shown). These results demonstrated that reovirusreplication in these animals was highly tumor specific with viralamplification only in the target U-87 cells.

Since most tumors are highly vascularized, it was likely that some viruscould enter the blood stream following the lysis of the infected tumorcells. To determine if there was systemic spread of the virus, blood washarvested from the treated and control animals, and serially diluted forsubsequent plaque titration. Infectious virus was found to be present inthe blood at a concentration of 1×10⁵ PFUs/ml (data not shown).

The high degree of tumor specificity of the virus, combined withsystemic spread, suggested that reovirus could be able to replicate inglioblastoma tumors remote from the initially infected tumor, asdemonstrated above with regard to breast cancer cells. To verify thishypothesis, SCID mice were implanted bilaterally with U-87 human tumorxenografts on sites overlying each hind flank of the animals. Thesetumors were allowed to grow until they measured 0.5×0.5 cm. Theleft-side tumors were then injected with a single dose (1×10⁷ pfu) ofreovirus in treated animals (n=5); control animals (n=7) weremock-treated with UV-inactivated virus. Tumors were again measured twiceweekly for a period of four weeks.

Results, shown in FIG. 4, demonstrated that inhibition and eventualregression of both the treated/injected (circles) and untreated tumormasses (squares) occurred only in the live reovirus-treated animals(open circles and squares), in contrast with the inactivatedreovirus-treated animals (closed circles and squares). Subsequentimmunofluorescent analysis revealed that reovirus proteins were presentin both the ipsilateral (treated) as well as the contralateral(untreated) tumor, indicating that regression on the untreated side wasa result of reovirus oncolysis (data not shown).

Pancreatic Carcinoma

Cell lines derived from pancreatic cancer were investigated for theirsusceptibility to reovirus infection. The cell lines included Capan-1(ATCC deposit HTB-79), BxPC3 (ATCC deposit CRL-1687), MIAPACA-2 (ATCCdeposit CRL-1420), PANC-1 (ATCC deposit CRL-1469), AsPC-1 (ATCC depositCRL-1682) and Hs766T (ATCC deposit HTB-134).

Five of these six cell lines demonstrated susceptibility to reovirusinfection including Capan-1, MIAPACA-2, PANC-1, AsPC-1 and Hs766T,whereas BxPC3 demonstrated little infectability as assayed byvirus-induced cytopathological effects, immunofluorescence and[³⁵S]-labelling. Interestingly, four of the five cell linesdemonstrating susceptibility to reovirus oncolysis have been shown topossess transforming mutations in codon 12 of the K-ras gene (Capan-1,MIAPACA-2, PANC-1 and AsPC-1) whereas the one lacking suchsusceptibility (BxPC3) has been shown to lack such a mutation (Berrozpe,G., et al. (1994), Int. J. Cancer, 58:185-191). The status of the otherK-ras codons is currently unknown for the Hs766T cell line.

Example 12 Use of Reovirus as an Oncolytic Agent in Immune-CompetentAnimals

A syngeneic mouse model was developed to investigate use of reovirus inimmune-competent animals rather than in SCID mice as described above.C3H mice (Charles River) were implanted subcutaneously with 1.0×10⁶ PFUsras-transformed C3H cells (a gift of D. Edwards, University of Calgary).Following tumor establishment, mice were treated with a series ofintratumoral injections of either live reovirus (1.0×10⁸ PFUs) orUV-inactivated reovirus. Following an initial series (six injectionsover a nine-day course), test animals received a treatment of dilutereovirus (1.0×10⁷ PFUs) every second day. Mock-treated animals receivedan equivalent amount of UV-inactivated virus.

FIG. 5 demonstrates that reovirus was an effective oncolytic agent inthese immune competent animals. All of the test animals showedregression of tumors; 5 of the 9 test animals exhibited complete tumorregression after 22 days, a point at which the control animals exceededacceptable tumor burden. Furthermore, there were no identifiable sideeffects in the animals treated with reovirus.

To assess the effects of previous reovirus exposure on tumor repressionand regression, one-half of a test group was challenged with reovirus(intramuscular injection of 1.0×10⁸ PFUs, type 3 Dearing) prior to tumorestablishment. Two weeks after challenge, neutralizing antibodies couldbe detected in all exposed animals. Following tumor establishment,animals were treated with a series of intratumoral injections of eitherlive or UV-inactivated reovirus, as described above.

FIG. 6 demonstrates that animals with circulating neutralizingantibodies to reovirus (i.e., those challenged with reovirus prior totumor establishment) exhibited tumor repression and regression similarto those animals in which there was no prior exposure to reovirus. Thus,reovirus can serve as an effective oncolytic agent even inimmune-competent animals with previous exposure to reovirus.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A method of treating a ras-mediated neoplasm in a human, comprising:(a) administering to the human an effective amount of a reovirus toresult in substantial oncolysis of the cells of the ras-mediatedneoplasm; and (b) administering to the human an effective amount of achemotherapeutic agent.
 2. The method of claim 1, wherein step (a) isperformed prior to step (b).
 3. The method of claim 1, wherein step (b)is performed prior to step (a).
 4. The method of claim 1, wherein thereovirus is a mammalian reovirus.
 5. The method of claim 4, wherein themammalian reovirus is a human reovirus.
 6. The method of claim 1,wherein the reovirus is selected from the group consisting of serotype 1reovirus, serotype 2 reovirus and serotype 3 reovirus.
 7. The method ofclaim 1, wherein more than one dose of reovirus is administered.
 8. Themethod of 1, wherein the reovirus is administered by injection into ornear the ras-mediated neoplasm.
 9. The method of claim 1, wherein theeffective amount of the reovirus is from about 10² pfu/kg body weight toabout 10¹³ pfu/kg body weight.
 10. The method of claim 1, wherein thechemotherapeutic agent is selected from the group consisting of5-fluorouracil, mitomycin C, methotrexate, hydroxyurea,cyclophosphamide, dacarbazine, mitoxantrone, epirubicin, doxorubicin,herceptin, etoposide, pregnasome, carboplatin, cisplatin, taxol,taxotere, tamoxifen, anti-estrogens, interferons, aromatase inhibitors,progestational agents and LHRH analogs.
 11. The method of claim 1,wherein the chemotherapeutic agent is taxol.
 12. The method of claim 1,wherein the chemotherapeutic agent is cisplatin.
 13. The method of claim1, wherein the chemotherapeutic agent is cyclophosphamide.